This invention relates to relatively short peptides about 25-40 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogs to the naturally available peptides, and which include three cyclizing disulfide linkages and one or more xcex3-carboxyglutamate residues.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Mollusks of the genus Conus produce a venom that enables them to carry out their unique predatory lifestyle. Prey are immobilized by the venom that is injected by means of a highly specialized venom apparatus, a disposable hollow tooth that functions both in the manner of a harpoon and a hypodermic needle.
Few interactions between organisms are more striking than those between a venomous animal and its envenomated victim. Venom may be used as a primary weapon to capture prey or as a defense mechanism. Many of these venoms contain molecules directed to receptors and ion channels of neuromuscular systems.
The predatory cone snails (Conus) have developed a unique biological strategy. Their venom contains relatively small peptides that are targeted to various neuromuscular receptors and may be equivalent in their pharmacological diversity to the alkaloids of plants or secondary metabolites of microorganisms. Many of these peptides are among the smallest nucleic acid-encoded translation products having defined conformations, and as such, they are somewhat unusual. Peptides in this size range normally equilibrate among many conformations. Proteins having a fixed conformation are generally much larger.
The cone snails that produce these toxic peptides, which are generally referred to as conotoxins or conotoxin peptides, are a large genus of venomous gastropods comprising approximately 500 species. All cone snail species are predators that inject venom to capture prey, and the spectrum of animals that the genus as a whole can envenomate is broad. A wide variety of hunting strategies are used, however, every Conus species uses fundamentally the same basic pattern of envenomation.
Several peptides isolated from Conus venoms have been characterized. These include the xcex1-, xcexc- and xcfx89-conotoxins which target nicotinic acetylcholine receptors, muscle sodium channels, and neuronal calcium channels, respectively (Olivera et al., 1985). A conotoxin, TxVIIA, containing a xcex3-carboxyglutamate residue and three disulfide bonds has been isolated (Fainzilber et al., 1991). Conopressins, which are vasopressin analogs, have also been identified (Cruz et al., 1987). In addition, peptides named conantokins have been isolated from Conus geographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990). These peptides have unusual age-dependent physiological effects: they induce a sleep-like state in mice younger than two weeks and hyperactive behavior in mice older than 3 weeks (Haack et al., 1990). Recently, peptides named contryphans containing D-tryptophan or D-leucine residues have been isolated from Conus radiatus (U.S. Ser. No. 09/061,026), and bromo-tryptophan conopeptides have been isolated from Conus imperialis and Conus radiatus (U.S. Ser. No. 08/785,534).
Ion channels are integral plasma membrane proteins responsible for electrical activity in excitable tissues. It has been recognized that slow inward currents can influence neuronal excitability via long-lasting depolarizations of the cell membrane (Llinxc3xa1s, 1988). The role of slow inward currents in generating endogenous bursting behavior has been recognized in molluscan neurons (Wilson and Wachtel, 1974; Eckert and Lux, 1976; Partridge et al., 1979), and more recently in some types of mammalian neurons (Lanthorn et al., 1984; Stafstrom et al., 1985; Llinàs, 1988; Alonso and Llinàs, 1989). Changes in the slow inward currents carried by such nonspecific cation channels may play a crucial role in bursting and pacemaker activities in a variety of excitable systems, ranging from mammalian heart muscle to molluscan neurons (Partridge and Swandulla, 1988; Hoehn et al., 1993; Kits and Mansvelder, 1966; van Soest and Kits, 1997). Slow inward currents are also believed to be important in generating epileptiform bursting in regions of the brain such as the hippocampus.
It is desired to identify drugs which are useful for modulating slow inward cation channels in vertebrates involved in syndromes of clinical relevance, such as epileptic activity in hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart muscle (Reuter, 1984).
This invention relates to relatively short peptides about 25-40 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogs to the naturally available peptides, and which include three cyclizing disulfide linkages and one or more xcex3-carboxyglutamate residues.
More specifically, the present invention is directed to conopeptides having the general formula I:
Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Xaa3-Cys-Xaa2-Xaa2-Xaa2-Xaa2-Xaa4-Cys-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Cys-Xaa2-Xaa2-Xaa2-Xaa3-Xaa3-Xaa3-Cys-Xaa9-Xaa9-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10 (SEQ ID NO: 1), wherein Xaa1 is des-Xaa1 or any amino acid; Xaa2 is any amino acid; Xaa3 is des-Xaa3 or any amino acid; Xaa4 is Glu xcex3-Glu (xcex3-carboxyglutamic acid; also referred to as Gla) or Gln; Xaa5 is any amino acid; Xaa6 is any amino acid; Xaa7 is any amino acid; Xaa8 is des-Xaa8 or any amino acid; Xaa9 is des-Xaa9 or any amino acid; and Xaa10 is des-Xaa10 or any amino acid, with the provisos that (a) when all Xaa10 are des-Xaa10, then both Xaa9 are des-Xaa9 or any amino acid and (b) when all Xaa1 are des-Xaa1, then Xaa5-Xaa6-Xaa7-Xaa8- is not Ser-Asp-Asn.
general formula II:
Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Xaa3-Cys-Xaa2-Xaa2-Xaa2-Xaa2-Xaa4-Cys-Cys-Xaa5-Xaa6-Xaa7-Xaa8-Cys-Xaa2-Xaa2-Xaa2-Xaa3-Xaa3-Xaa3-Cys-Xaa9-Xaa9-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10-Xaa10 (SEQ ID NO:2), wherein Xaa1 is des-Xaa1 or any amino acid; Xaa2 is any amino acid; Xaa3 is des-Xaa3 or any amino acid; Xaa4 is Glu, xcex3-Glu or Gln; Xaa5 is Ser or Thr; Xaa6 is any amino acid; Xaa7 is any amino acid; Xaa8 is des-Xaa8 or any amino acid; Xaa9 is des-Xaa9 or any amino acid; and Xaa10 is des-Xaa10 or any amino acid, with the provisos that (a) when all Xaa10 are des-Xaa10, then both Xaa9are des-Xaa9 or any amino acid and (b) when all Xaa1 are des-Xaa1 and Xaa5 is Ser, then Xaa6-Xaa7-Xaa8- is not Asp-Asn.
general formula III:
Xaa1-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa2 (SEQ ID NO:3), wherein Xaa1 is any amino acid; Xaa2 is des-Xaa2 or any amino acid and Xaa3 is Glu or xcex3-Glu.
general formula IV:
Xaa1-Xaa2-Xaa2-Xaa2-Xaa2-Xaa2-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Xaa3-Xaa1-Xaa1-Xaa1-Xaa4-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa2 (SEQ ID NO:4), wherein Xaa1 is any amino acid; Xaa2 is des-Xaa2 or any amino acid; Xaa3 is Ser or Thr; and Xaa4 is Glu or xcex3-Glu.
or general formula V:
Xaa1-Xaa1-Xaa2-Cys-Xaa3-Xaa3-Xaa4-Phe-Xaa3-Xaa3-Cys-Thr-Xaa3-Xaa3-Ser-Xaa5-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa3-Leu-Xaa3-Xaa3-Xaa3-Xaa3-Xaa3 (SEQ ID NO:5), wherein Xaa1 is des-Xaa1 or any amino acid; Xaa2 is Asp, Glu or xcex3-Glu; Xaa3 is any amino acid; Xaa4 is Trp or 6-bromo-Trp; and Xaa5 is Glu or xcex3-Glu.
The amino acid or the amino acid residues of the peptides is an amino acid selected from the group consisting of natural, modified or non-natural amino acids. The disulfide bridges in the conopeptides of general formulas I-V (as well as the specific conopeptides described herein) are between the first and fourth cysteine residues, between the second and fifth cysteine residues and between the third and sixth cysteine residues. The C-terminal end may contain a carboxyl or amide group. The invention also includes pharmaceutically acceptable salts of the conopeptides. These conopeptides are useful for modulating slow inward cation channels in vertebrates involved in syndromes of clinical relevance, such as epileptic activity in hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart muscle (Reuter, 1984). Thus, the conopeptides are useful as agonists of neuronal pacemaker cation channels.
The invention further relates to the specific peptides:
Asp-Cys-Thr-Ser-Xaa1-Phe-Gly-Arg-Cys-Thr-Val-Asn-Ser-Xaa2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Xaa2-Leu-Tyr-Ala-Phe-Xaa3-Ser (SEQ ID NO:6) (PnVIIA), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or hydroxy-Pro (Hyp), preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Xaa1-Leu-Xaa2-Cys-Ser-Val-Xaa1-Phe-Ser-His-Cys-Thr-Lys-Asp-Ser-Xaa2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Gln-Thr-Tyr-Cys-Thr-Leu-Met-Xaa3-Xaa3-Asp-Xaa1 (SEQ ID NO:7) (Tx6.4), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Xaa1-Xaa1-Arg-Xaa1-Gly-Gly-Cys-Met-Ala-Xaa1-Phe-Gly-Leu-Cys-Ser-Arg-Asp-Ser-Xaa2-Cys-Cys-Ser-Asn-Ser-Cys-Asp-Val-Thr-Arg-Cys-Xaa2-Leu-Met-Xaa3-Phe-Xaa3-Xaa3-Asp-Xaa1 (SEQ ID NO:8) (Tx6.9), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa2-Ala-Asp-Ser-Xaa2-Cys-Cys-Thr-Xaa2-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:9) (J010), wherein Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; and the C-terminus is a free carboxyl group or is amidated, preferably amidated;
Asp-Xaa1-Xaa1-Asp-Asp-Gly-Cys-Ser-Val-Xaa1-Gly-Xaa3-Cys-Thr-Val-Asn-Ala-Xaa2-Cys-Cys-Ser-Gly-Asp-Cys-His-Xaa2-Thr-Cys-Ile-Phe-Gly-Xaa,-Xaa2-Val (SEQ ID NO:10) (Tx6.6), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Gly-Met-Xaa1-Gly-Xaa2-Cys-Lys-Asp-Gly-Leu-Thr-Thr-Cys-Leu-Ala-Xaa3-Ser-Xaa2-Cys-Cys-Ser-Xaa2-Asp-Cys-Xaa2-Gly-Ser-Cys-Thr-Met-Xaa1(SEQ ID NO:11) (Tx6.5), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Xaa2-Cys-Arg-Ala-Xaa1-Tyr-Ala-Xaa3-Cys-Ser-Xaa3-Gly-Ala-Gln-Cys-Cys-Ser-Leu-Leu-Met-Cys-Ser-Lys-Ala-Thr-Ser-Arg-Cys-Ile-Leu-Ala-Leu (SEQ ID NO:12) (Gm6.7), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably a free carboxyl group;
Asn-Gly-Gln-Cys-Xaa2-Asp-Val-Xaa1-Met-Xaa3-Cys-Thr-Ser-Asn-Xaa1-Xaa2-Cys-Cys-Ser-Leu-Asp-Cys-Xaa2-Met-Tyr-Cys-Thr-Gln-Ile (SEQ ID NO:13) (Mr6.1), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; Xaa3 is Pro or Hyp, preferably Hyp; and the C-terminus is a free carboxyl group or is amidated, preferably amidated;
Cys-Gly-Gly-Xaa1-Ser-Thr-Tyr-Cys-Xaa2-Val-Asp-Xaa2-Xaa2-Cys-Cys-Ser-Xaa2-Ser-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe (SEQ ID NO:14) (Mr6.2), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; and the C-terminus is a free carboxyl group or is amidated, preferably amidated;
Asn-Gly-Gly-Cys-Lys-Ala-Thr-Xaa1-Met-Ser-Cys-Ser-Ser-Gly-Xaa1-Xaa2-Cys-Cys-Ser-Met-Ser-Cys-Asp-Met-Try-Cys (SEQ ID NO:15) (Mr6.3), wherein Xaa1 is Trp or 6-bromo-Trp; Xaa2 is Glu or xcex3-Glu, preferably xcex3-Glu; and the C-terminus is a free carboxyl group or is amidated, preferably amidated.
Finally, the invention further relates to the propeptide sequences for the above peptides and the DNA sequences coding for these propeptide sequences as described in further detail herein.
SEQ ID NO:1=xcex3-conopeptides of general formula I; SEQ ID NO:2=xcex3-conopeptides of general formula II; SEQ ID NO:3=xcex3-conopeptides of general formula III; SEQ ID NO:4=xcex3-conopeptides of general formula IV; SEQ ID NO:5=xcex3-conopeptides of general formula V; SEQ ID NO:6=xcex3-conopeptide corresponding to PnVIIA; SEQ ID NO:7=xcex3-conopeptide corresponding to Tx6.4; SEQ ID NO:8=xcex3-conopeptide corresponding to Tx6.9; SEQ ID NO:9=xcex3-conopeptide corresponding to J010; SEQ ID NO:10=xcex3-conopeptide corresponding to Tx6.6; SEQ ID NO:11=xcex3-conopeptide corresponding to Tx6.5; SEQ ID NO:12=xcex3-conopeptide corresponding to Gm6.7; SEQ ID NO:13=xcex3-conopeptide corresponding to Mr6.1; SEQ ID NO:14=xcex3-conopeptide corresponding to Mr6.2; SEQ ID NO:15=xcex3-conopeptide corresponding to Mr6.3; SEQ ID NO:16=DNA encoding propeptide of Tx6.4; SEQ ID NO:17=propeptide of Tx6.4; SEQ ID NO:18=DNA encoding propeptide of Tx6.9; SEQ ID NO:19=propeptide of Tx6.9; SEQ ID NO:20=DNA encoding propeptide of J010; SEQ ID NO:21=propeptide of J010; SEQ ID NO:22=DNA encoding propeptide of Tx6.6; SEQ ID NO:23=propeptide of Tx6.6; SEQ ID NO:24=DNA encoding propeptide of Tx6.5; SEQ ID NO:25=propeptide of Tx6.5; SEQ ID NO:26=DNA encoding propeptide of Gm6.7; SEQ ID NO:27=propeptide of Gm6.7; SEQ ID NO:28=DNA encoding propeptide of Mr6. 1; SEQ ID NO:29=propeptide of Mr6.1; SEQ ID NO:30=DNA encoding propeptide of Mr6.2; SEQ ID NO:31=propeptide of Mr6.2; SEQ ID NO:32=DNA encoding propeptide of Mr6.3; SEQ ID NO:33=propeptide of Mr6.3; SEQ ID NO:34=DNA encoding propeptide of Tx6.1; SEQ ID NO:35=propeptide of Tx6.1; SEQ ID NO:36=xcex3-conopeptide corresponding to Tx6.1; SEQ ID NO:37=consensus sequence of xcex3-conopeptides PnVIIA and Tx6.4; SEQ ID NO:38=degenerate probe for consensus sequence of xcex3-conopeptides; SEQ ID NO:39=degenerate probe for consensus sequence of xcex3-conopeptides; SEQ ID NO:40=consensus sequence of pro-xcex3-conopeptides; SEQ ID NO:41=degenerate probe for consensus sequence of pro-xcex3-conopeptides; SEQ ID NO:42=xcex3-conopeptide PnVIIA; SEQ ID NO:43=xcex3-conopeptide TxVIIA; SEQ ID NO:44=N-terminal tryptic peptide of xcex3-conopeptide PnVIIA; SEQ ID NO:45=C-terminal tryptic peptide of xcex3-conopeptide PnVIIA; SEQ ID NO:46=primer for isolating conopeptides from Conus textile cDNA library; SEQ ID NO:47=primer for isolating conopeptides from Conus textile cDNA library.
This invention relates to relatively short peptides about 25-40 residues in length, which are naturally available in minute amounts in the venom of the cone snails or analogs to the naturally available peptides, and which include three cyclizing disulfide linkages and one or more xcex3-carboxyglutamate residues.
More specifically, the present invention is directed to conopeptides having the general formulas I-V described above. The invention is also directed to the specific xcex3-conopeptides PnVIIA, Tx6.4, Tx6.9, J010, Tx6.6, Tx6.5, Gm6.7, Mr6.1, Mr6.2 and Mr6.3, the sequences of which are described above.
The invention is further directed to isolated nucleic acids which encode xcex3-conopeptides, including the above and xcex3-conopeptide Tx6.1, and to isolated propeptides encoded by the nucleic acids. This aspect of the present invention is set forth in Table 1.
The conopeptides of the present invention are useful for modulating slow inward cation channels in vertebrates involved in syndromes of clinical relevance, such as epileptic activity in hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart muscle (Reuter, 1984). Thus, the conopeptides are useful as agonists of neuronal pacemaker cation channels.
The xcex3-conopeptides of the present invention are identified by isolation from Conus venom. Alternatively, the xcex3-conopeptides of the present invention are identified using recombinant DNA techniques. According to this method of identification, cDNA libraries of various Conus species are screened using conventional techniques with degenerate probes for the peptide consensus sequence Xaa-Cys-Cys-Ser (SEQ ID NO:37), wherein Xaa is Glu or Gln. Suitable probes are 5xe2x80x2 SARTGYTGYAGY 3xe2x80x2 (SEQ ID NO:38) or 5xe2x80x2 SARTGYTGYTCN 3xe2x80x2 (SEQ ID NO:39). Alternatively, cDNA libraries are screened with degenerate probes for the propeptide consensus sequence Ile-Leu-Leu-Val-Ala-Ala-Val-Leu (SEQ ID NO:40). Suitable probes for this sequence are 5xe2x80x2 ATHYTNYTNGTNGCNGCNGTNYTN 3xe2x80x2 (SEQ ID NO:4 1). Clones which hybridize to these probes are analyzed to identify those which meet minimal size requirements, i.e., clones having approximately 300 nucleotides (for a propeptide), as determined using PCR primers which flank the cDNA cloning sites for the specific cDNA library being examined. These minimal-sized clones are then sequenced. The sequences are then examined for the presence of a peptide having the characteristics noted above for xcex3-conopeptides, such as the presence of a Glu residue which could be modified to a xcex3-Glu and 6 cysteine residues. The biological activity of the peptides identified by this method is tested as described herein.
These peptides are sufficiently small to be chemically synthesized. General chemical syntheses for preparing the foregoing conopeptides peptides are described hereinafter, along with specific chemical synthesis of conopeptides and indications of biological activities of these synthetic products. Various ones of these conopeptides can also be obtained by isolation and purification from specific Conus species using the techniques described in U.S. Pat. No. 4,447,356 (Olivera et al., 1984), U.S. Pat. No. 5,514,774 (Olivera et al., 1996) and U.S. Pat. No. 5,591,821 (Olivera et al., 1997), the disclosures of which are incorporated herein by reference.
Although the conopeptides of the present invention can be obtained by purification from cone snails, because the amounts of conopeptides obtainable from individual snails are very small, the desired substantially pure conopeptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy. For example, the yield from a single cone snail may be about 10 micrograms or less of conopeptide. By xe2x80x9csubstantially purexe2x80x9d is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity. Chemical synthesis of biologically active conopeptides depends of course upon correct determination of the amino acid sequence. Thus, the conopeptides of the present invention may be isolated, synthesized and/or substantially pure.
The conopeptides can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1979). The peptides produced in this manner are isolated, reduced if necessary, and oxidized to form the correct disulfide bonds, if present in the final molecule.
One method of forming disulfide bonds in the conopeptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides. The oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. It is also found that the linear peptide, or the oxidized product having more than one fraction, can sometimes be used for in vivo administration because the cross-linking and/or rearrangement which occurs in vivo has been found to create the biologically potent conopeptide molecule. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence. Use of various coupling reagents, e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole, various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavage reagents, to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology. Classical solution synthesis is described in detail in the treatise, xe2x80x9cMethoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden,xe2x80x9d (1974). Techniques of exclusively solid-phase synthesis are set forth in the textbook, xe2x80x9cSolid-Phase Peptide Synthesis,xe2x80x9d (Stewart and Young, 1969), and are exemplified by the disclosure of U.S. Pat. No. 4,105,603 (Vale et al., 1978). The fragment condensation method of synthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Other available syntheses are exemplified by U.S. Pat. No. 3,842,067 (1974) and U.S. Pat. No. 3,862,925 (1975). The synthesis of peptides containing xcex3-carboxyglutamic acid residues is exemplified by Rivier et al. 29 (1987), Nishiuchi et al. (1993) and Zhou et al. (1996). Synthesis of conopeptides have been described in U.S. Pat. No. 4,447,356 (Olivera et al., 1984), U.S. Pat. No. 5,514,774 (Olivera et al., 1996) and U.S. Pat. No. 5,591,821 (Olivera et al., 1997).
Common to such chemical syntheses is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an xcex1-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the xcex1-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in such a synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side-chain protecting groups linked to various ones of the residues having labile side chains.
As far as the selection of a side chain amino protecting group is concerned, generally one is chosen which is not removed during deprotection of the xcex1-amino groups during the synthesis. However, for some amino acids, e.g., His, protection is not generally necessary. In selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following general rules are followed: (a) the protecting group preferably retains its protecting properties and is not split off under coupling conditions, (b) the protecting group should be stable under the reaction conditions selected for removing the xcex1-amino protecting group at each step of the synthesis, and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
It should be possible to prepare many, or even all, of these peptides using recombinant DNA technology. However, when peptides are not so prepared, they are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected xcex1-amino acid to a suitable resin. Such a starting material can be prepared by attaching an xcex1-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine (MBHA) resin. Preparation of the hydroxymethyl resin is described by Bodansky et al. (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, Calif.) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart and Young (1969). BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus. Thus, solid resin supports may be any of those known in the art, such as one having the formulae xe2x80x94Oxe2x80x94CH2-resin support, xe2x80x94NH BHA resin support, or xe2x80x94NH-MBHA resin support. When the unsubstituted amide is desired, use of a BHA or MBHA resin is preferred, because cleavage directly gives the amide. In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should still other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text (1974).
The C-terminal amino acid, protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Horiki et al. (1978), using KF in DMF at about 60xc2x0 C. for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized. Following the coupling of the BOC-protected amino acid to the resin support, the xcex1-amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The deprotection is carried out at a temperature between about 0xc2x0 C. and room temperature. Other standard cleaving reagents, such as HCl in dioxane, and conditions for removal of specific xcex1-amino protecting groups may be used as described in Schroder and Lubke (1965).
After removal of the xcex1-amino-protecting group, the remaining xcex1-amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. Selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).
The activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,Nxe2x80x2-diisopropylcarbodiimide and N-ethyl-Nxe2x80x2-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder and Lubke (1965) and Kapoor (1970).
Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH2Cl2 (1:1) or in DMF or CH2Cl, alone. In cases where intermediate coupling occurs, the coupling procedure is repeated before removal of the xcex1-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis, if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970). Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978).
After the desired amino acid sequence has been completed, the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the xcex1-amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid. If Met is present in the sequence, the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation. When using hydrogen fluoride or TFA for cleaving, one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues. To effect such a disulfide cyclizing linkage, fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected. Alternatively, deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0xc2x0 C. with hydrofluoric acid (HF) or TFA, followed by oxidation as described above. A suitable method for cyclization is the method described by Cartier et al. (1996).
The present xcex3-conotoxins are useful for modulating slow inward cation channels in 1:57, vertebrates involved in syndromes of clinical relevance, such as epileptic activity in hippocampus (Hoehn et al., 1993) and pacemaker potentials in heart muscle (Reuter, 1984). Thus, the conopeptides are useful as agonists of neuronal pacemaker cation channels.
Pharmaceutical compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington""s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, an antagonistic amount of active ingredient will be admixed with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral or parenteral. The compositions may further contain antioxidizing agents, stabilizing agents, preservatives and the like.
For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution of a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
The conopeptides are administered in an amount sufficient to agonize the neuronal pacemaker calcium channels. The dosage range at which the conopeptides exhibit this agonistic effect can vary widely depending upon the particular condition being treated, the severity of the patient""s condition, the patient, the specific conopeptide being administered, the route of administration and the presence of other underlying disease states within the patient. Typically, the conopeptides of the present invention exhibit their therapeutic effect at a dosage range from about 0.05 mg/kg to about 250 mg/kg, and preferably from about 0.1 mg/kg to about 100 mg/kg of the active ingredient. A suitable dose can be administered in multiple sub-doses per day. Typically, a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form. A more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form. Dosages are generally initiated at lower levels and increased until desired effects are achieved.